Changes in lipids metabolism indices as a result of different form of selenium supplementation in chickens

Selenium is an essential element that is important for many metabolic processes. Feed components used in chicken nutrition, especially cereals, may be deficient in selenium, hence selenium supplementation is necessary. Taking into account the progress in breeding, and thus the higher demand of birds for this element, it seems obvious to investigate an increased selenium dose in the diet of chickens. The aim of the study was to evaluate the effect of feed enriched with different forms of selenium at an increased dose of 0.5 mg/kg feed on the profile and metabolism of fatty acids in the breast muscle and liver of chickens. The study was conducted on 300 Ross 308 chickens reared for 42 days under standard conditions. The control group received feed supplemented with sodium selenite at a dose of 0.3 mg/kg feed. The research groups received different forms of selenium (sodium selenate, selenised yeast, nano-selenium) at an increased dose of 0.5 mg/kg feed. The study showed that the administration of different forms of selenium in the feed affected its concentration in the breast muscle and liver (p ≤ 0.01). Nano-selenium was found to have a high bioavailability, but also a lower risk of toxicity compared to other forms of selenium. Using different forms of selenium (p ≤ 0.01) at a dose of 0.5 mg/kg feed can significantly modify the fatty acid profile, lipid and enzymatic indices of fatty acid metabolism in breast muscle and liver.

In monogastric animals, unlike in the ruminants, unsaturated fatty acids (UFA) are not biohydrogenated from feed. For this reason, there is a correlation between delivered dietary fatty acids (FA) and those deposited in tissues of such animals as poultry 1,2 . A high polyunsaturated FA (PUFA) concentration in the feedstuff bears the risk of increased susceptibility of tissue lipids to oxidation, which in turn may modify both taste and aroma (rancid) and the nutritive value of meat. To prevent oxidative processes, feed mixtures for poultry are supplemented with additives featuring antioxidative properties that preserve oxidative stability and, by this means, also the oxidative balance of lipids in meat 3 . Selenium (Se) is one of the commonly applied antioxidants. It is used in animal feeding usually in two forms: inorganic-as sodium selenite (Na 2 SeO 3 ) or sodium selenate (Na 2 SeO 4 ; SS), or organic-as selenomethionine (SeMet) or selenocysteine (SeCys). Selenium is an essential bioelement which-together with other microelements, like zinc and iodine-plays an important role in the proper functioning, development, and growth of various organisms 4 . So far, feed mixtures for broiler chickens have mainly been supplemented with inorganic forms of Se. However, with the advance of research on Se, also its other forms have been implemented, including especially these more readily absorbed in tissues compared to SS and sodium selenate 5 . The Se mitigate oxidative stress and peroxidative damage of UFA, and affect the effectiveness of fatty acid biosynthesis in animal tissues 6,7 . Selenium deficiency in diet may adversely influence the conversion of linolenic acid (ALA) to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), resulting in the unbeneficial n-6/n-3 ratio in tissue lipids. Also, Se addition to feedstuffs for livestock modifies the fatty acid profile of meat [8][9][10][11][12] . The consumption of chicken meat increases successively each year compared to other meat types. Therefore, the enrichment of diets for poultry with appropriate forms of Se would allow producing poultry meat rich in high-quality n-3 fatty acids that are indispensable in the human diet. Recent studies have indicated that especially the long-chain n-3
This study analyzed feed mixtures supplemented with various forms of Se in a dose of 0.5 mg/kg feed, which is upper limits of organic Se supplementation in the chicken feed. A comparison of the fatty acid profile of BM intramuscular fat and liver fat of birds from the particular experimental groups demonstrated that the administration of the increased doses of various Se forms significantly (p ≤ 0.01) affected the lipid composition of the examined tissues, including concentrations of SFA, MUFA, PUFA as well as n-3 and n-6 FA ( Table 1). The major FA responsible for differences in the content of PUFA was C18:2 acid (LA) in BM samples and C20:4 acid (n-6) in liver samples. The use of SY or nano-Se in chicken feeding enables significantly increasing MUFA contents and increasing PUFA contents in BM The highest content of n-3 FA in the samples of BM intramuscular fat was  www.nature.com/scientificreports/ determined in group T1, where birds received a diet with an increased SS dose. The increased content of n-3 fatty acids resulted in the lowest content of n-6 fatty acids and a lower n-6/n-3 ratio. In the case of the liver fat samples, the most beneficial n-6/n-3 ratio was determined in the CON group, which additionally featured the highest content of n-3 fatty acids. Broiler diet supplementation with SY and nano-Se significantly (p ≤ 0.01) increased PUFA content in BM intramuscular fat. In the case of liver fat samples, the same effect (p ≤ 0.01) was achieved upon diet supplementation with an Se dose of 0.3 mg/kg feed. The contents of LA and ALA fatty acids, which are significantly depended on Se form used in the broiler diet. The highest LA content in BM was determined in group T2, whereas the lowest one in group T1. The ALA content was inversely proportional to the LA content. The highest ALA content was determined in group T1 fed a diet with the increased SS dose, whereas the lowest one in groups T2 and T3. The analysis of DHA contents in BM and liver demonstrated their significantly (p ≤ 0.01) higher content in the groups of chicken broilers administered the SS-supplemented diet. The highest Se concentration in the breast muscle was determined in group T2 (p ≤ 0.01) fed a diet with SY (Table 2), whereas the lowest one in BM of the broilers from group T3. In the case of the liver samples, the highest Se concentration was determined in the CON group fed a diet supplemented with the recommended SS dose (0.3 mg Se/kg feed). The analysis of lipid oxidation indices showed significant (p ≤ 0.01) differences in their values depending on Se form. The TBA was significantly higher in the groups fed diets with SS (CON and T1), whereas the total antioxidative capacity measured with the radical scavenging method (DPPH, 1,1-diphenyl-2-picrylhydrazyl) and GSH was significantly higher in BM and liver of birds from groups T2 and T3.
The form of Se used in the diet for broilers had no significant (p ≥ 0.05) effect on PUFA/SFA ratio in BM, whereas its values in liver samples differed significantly (p ≤ 0.01) among the groups, with the highest PUFA:SFA ratio found in group CON ( Table 3). The n-6/n-3 ratio was significantly higher in groups CON and T1, regardless of tissue examined. Fat from BM and liver of birds from groups and T featured a significantly (p ≤ 0.01) lower AI value, whereas BM fat from groups CON, T2, and T3, and liver fat from group T1 had a significantly (p ≤ 0.01) lower TI value. Sodium selenate administered to birds in the higher dose tested (0.5 mg/kg feed, group T1) had a negative effect on the TI value in BM, whereas in liver samples the same effect was observed upon diet supplementation with nano-Se. The values of hypocholesterolemic and hypercholesterolemic indices (h/H) were significantly (p ≤ 0.01) higher in the groups fed the SS-supplemented diet, both in breast muscle and liver. In addition, the dietary form of Se had a significant (p ≤ 0.01) effect on the values of DI (18), DI (16), total DI, thioesterase index (only in BM) and activity of Δ5-desaturase + Δ6-desaturase. The elongase activity (EI) differed significantly (p ≤ 0.05) between the groups; with the highest activity determined in both BM and liver of the birds from the groups fed the SS-supplemented diet. Contents of neutral and hypocholesterolemic acids (DFA) differed significantly between the tissues examined. The highest DFA concentration was determined in both BM and liver of the control birds. The value of the saturation index (S/P) and the content of hypercholesterolemic FA (HFA) differed significantly (p ≤ 0.01). The lowest values of these parameters were determined in the groups of birds fed the SS-supplemented diet.

Discussion
Selenium is an essential element playing an important role in many physiological processes in animal bodies. It affects the appropriate growth, fertility, and immune responses 21 . In birds, Se is indispensable for the synthesis of selenocysteine, i.e. an amino acid being a constituent of selenoproteins involved in metabolic processes, like glutathione peroxidase (GSH-Px), thioredoxin reductase, and iodothyronine deiodinase 22 . Dietary components used in broiler feeding, including mainly cereals, can be deficient in Se mainly due to its shortages in the soil. Therefore, Se is supplemented in the poultry production to prevent its deficiencies in broiler chicken diet 23,24 . Broiler body demand for Se has been established at 0.15 mg/kg feed 25 . In turn, other authors recommend increasing this dose to 0.3 and 0.75 mg/ kg feed 26,27 . Given the ever increase rate of broiler chicken growth and, consequently, their faster body metabolism, it seems advisable to examine the impact of an increased diet supplementation with Se on their body protection against reactive oxygen species. This would help providing consumers with high-quality meat rich in fine-quality fat featuring a beneficial fatty acid profile 27,28 . Supplementation of a diet for broilers chicken with Se affects its content in meat 29 , whereas Se form influences also its absorption, retention, and utilization in the bird bodies 30 . Selenium selenate is absorbed by the body via simple diffusion, whereas SY-via the active transport. This allows the birds to accumulate Se in tissues and then to use it for oxidative defense in the instance of intensified stress 31 . In the study by 29 , the highest Se concentration in BM and liver tissues was determined in the birds fed the SY-supplemented diet. These authors also showed that organic Se was more readily absorbed by the body compared to inorganic Se. In turn, in the present study, the highest Se concentration in BM was achieved in the birds from group T2 fed a diet with an increased dose of SY, whereas the highest Se concentration in the liver was determined in the birds from group T1 (SS, 0.5 mg/ kg). This result also contradicts findings reported by 32 . The conducted study confirms that nano-Se shows higher bioavailability and lower toxicity risk than other Se forms 33 , especially SS. In view of the results of the present study, the recommendations for broiler diet supplementation with nano-Se at 0.3-0.5 mg/kg seem justified 26 . Furthermore, nano-Se added to a broiler diet in a upper recommended amount (0.5 mg/kg) may aid the proper functioning of the gastrointestinal tract. By this means, it can affect the proper development of birds' immunity 34 , ensuring their high welfare and top quality of the finished product.
Given the increased risk of toxicity of inorganic forms due to their easy accumulation in tissues, there is a need for research on other forms of Se, which will provide important information for modern poultry production for the sake of both chickens welfare food safety for humans. 100 g of breast muscle from chickens fed a diet with elevated levels of nano-Se in feed (0.5 mg/kg) can provide 17% of the Recommended Daily Intake (RDI of Se = 70 µg/day) for an adult human 35 . Consumption of breast muscle from T2 chickens provides up to 60% of the RDI. In contrast, Se tends to concentrate strongly in the liver. Livers from chickens fed CON, T1, (inorganic Se) diets had a successively four, five times higher recommended RDI, while those from the T2 group had more than one time higher RDI, which may pose a risk to human health. Livers from T2 chickens could potentially provide up to 70% of the RDI of this element (per 100 g of chicken liver). Appropriate supplementation with nano-Se at an increased dose (0.5 mg/kg) can result in a final product with increased nutritional value, referred to as functional food. This confirms that meat and livers enriched with nano-Se in this manner are a valuable  www.nature.com/scientificreports/ and safe source of Se that can contribute to improved consumer health, while maintaining the need for this element in broiler chickens and ensuring their normal growth, health and thus welfare levels. The use of nano-Se in the diet of chickens at the maximum dose of 0.5 mg/kg is allowed by the European Union regulations 36 , as it brings mutual benefits for both the chickens and the quality of the raw material obtained from the birds. Also, nano-Se seems effective in modulating the fatty acid profile of broiler chicken meat and giblets. These findings indicate that the supplementation of broiler diet with the nanosized form of Se at the dose as in the present study is safe as its other forms in terms of enriching meat with Se, which may be important for the consumer health. In addition, nano-Se protects the liver of broiler chickens against the detrimental effect of Cr(VI) 37 . In their study 38 supplemented a broiler diet with SY in a dose of 0.5 mg/kg and found that it allowed producing meat enriched with selenium at 0.256 mg/kg and enriched with n-3 FA up to 6.71% of total FA. The comparison of results obtained for the SY-supplemented group demonstrated that it was characterized by the highest Se concentration in BM (0.42 mg/kg) and by a substantially lower content of the n-3 fatty acids (1.54% of total FA). The analysis of the present study results in terms of the human diet recommendation shows that supplementing broiler diet with a higher SS dose (0.5 mg/kg feed) allows achieving the highest Se concentration in BM, the best n-6/n-3 ratio, and the highest ALA concentration. Broiler diet supplementation with SS resulted in lower contents of C14:0 and C:16 in BM and in the liver, compared to the other additives tested. The above findings are essential from the standpoint of human nutrition because these FA can be potential promoters of cardiovascular diseases 39 . Broiler diet supplementation with SS (groups CON and T1) caused also a clear tendency for the increased concentration of n-3 PUFAs, compared to the other Se forms tested. This correlation can be explained by the greater protection ensured by inorganic Se and, thus, by reduced degradation of PUFAs in oxidative processes 40 . The latter can be ascribed to the activity of glutathione peroxidase (GPx), i.e. a selenium-dependent enzyme the main function of which is to eliminate free radicals and, consequently, to protect FA, including especially PUFAs. Selenium is an important component of GSH-Px, whereas Se supplementation can enhance mRNA GSH-Px1 expression in the liver of chickens 41 . In the present study, Se used in the form of SY and nano-Se was found to significantly affect PUFA accumulation in BM, and to diminish it in the liver. This is linked to the functions of BM and liver. Breast muscle accumulates PUFA mainly in lipids whereas liver is responsible for their distribution in the body. The increased level of PUFA in a diet, especially of n-3 PUFA, can lead to lipid peroxidation and, consequently, to the impairment of liver functions 42,43 . We evidenced that the use of other than SS forms of Se had a more beneficial effect on the proper lipid metabolism in birds.
The n-3 and n-6 FA belong to two different families and cannot be synthesized by the organisms of mammals and birds. The excess of fatty acids from one family impairs the metabolism of those from the other group; hence, it is essential to maintain the optimal n-6/n-3 ratio. The use of SS in the chickens diet in the present study was found to significantly modify the FA composition, by ensuring more beneficial n-6/n-3 ratio, which is recommended in the prevention of the ischemic heart disease 44 . The disturbed n-6/n-3 ratio leads to the overproduction of proinflammatory eicosanoids, which stimulate the synthesis of cytokines and acute phase proteins, which in turn are triggers of such diseases as neoplasms, cardiovascular diseases, atherosclerosis, obesity, type 2 diabetes, or Alzheimer's disease 45,46 . In turn, lauric (C12:0), myristic (C14:0), and palmitic (C16:0) acids strongly correlate with the risk of the incidence of atherosclerosis, obesity, or ischemic heart disease 47,48 , whereas the degree of saturation with these acids is considered to be a parameter of food quality assessment 49 . In the present study, dietary supplementation with SS significantly (p ≤ 0.01) diminished the content of saturated fatty acids and increased ALA content in BM, which is deemed important from the human nutrition perspective.
The TBARS value (mg MDA/kg sample) indicates the status of lipid oxidation in different tissues, whereas DPPH serves to measure the capability for scavenging free radicals 50,51 . The values of atherogenicity (AI) and thrombogenicity (IT) indices ought to be as low as possible. The lower their values are, the smaller is the probability of atherosclerosis incidence and blood clots development in humans 52 .
Unlike the AI and TI indices, the values of the hypocholesterolemic and hypercholesterolemic indices (h/H) need to be as high as possible to protect a consumer against hypercholesterolemia, which is a risk factor of the atherosclerotic syndrome 53 . In the present study, liver samples, having high contents of lipids and minerals that elicit prooxidative effects, had higher TBARS values compared to BM The addition of selenates (0.3 mg/kg) and nano-Se (0.5 mg/kg) to diet caused a significant (p ≤ 0.05) decrease in the content of lipid oxidation products in liver tissues, i.e. to 2.08 and 2.04 μg MDA/g, respectively. Also 54 observed the protective effect of Se on liver tissues against changes triggered by free radicals. They showed that diet supplementation with nano-Se significantly reduced the content of lipid oxidation products, from 0.55 in group C to 0.30 mg MDA/g in the amended group.
Delta-9 desaturase catalyzes the transformation of medium-chain and long-chain SFA into individual MUFA, i.e. C16:0 and C18:0 as well as C16:1 and C18:1, respectively 55 . Delta-9 desaturase activity depends on the diet and age of birds 56 , but also on the form of Se implemented in the diet, as indicated in the present study. The increase in delta-9 desaturase activity in the groups fed the SS-supplemented diet caused a decrease in contents of C16:0 and C18:0 FA in favor of C16:1 and C18:1 fatty acids in BM The organisms of both mammals and birds are incapable of synthesizing essential PUFA, like LA and ALA from acetyl-CoA, but can transform them into more unsaturated long-chain essential FA when they are provided with diet. Transformations of these acids are catalyzed by, among other things, desaturases. The Δ5 + Δ6-desaturase index is used to assess birds' capability to synthesize long-chain FA from LA and ALA 57 . The higher value of the Δ5 + Δ6-desaturase index indicates a higher effectiveness of long-chain FA synthesis. Broiler chickens are capable of synthesizing DHA and EPA from ALA, and this synthesis is catalyzed by desaturase and elongase 58 . In the present study, an increased DHA content was determined in both BM and liver of the birds fed the SS-supplemented diet. The EPA content did not differ significantly (p ≥ 0.05) between BM samples, but significant (p ≤ 0.01) differences were noted in its content between the liver samples. The increased contents of EPA and DHA are confirmed by a significantly www.nature.com/scientificreports/ higher activity of Δ5 + Δ6-desaturase in groups CON and T, which allows concluding that Se addition in the form of SS had a significant effect on the effectiveness of long-chain FA synthesis.

Conclusion
The study showed that selenium supplementation at a dose of 0.5 mg/kg feed significantly influences selenium concentration in breast muscle and liver, modifications of fatty acid profile, oxidative indices and lipid metabolism. The application of nano-Se in the amount of 0.5 mg/kg of feed is characterized by a significantly higher content of PUFAs and protection of lipids against the action of reactive oxygen species, with its high bioavailability and low toxicity for the chicken organism. The use of increased doses of selenium in feed is a response to the increasingly rapid growth rate and orgasmic metabolism of chickens. This will help to provide consumers with a high quality product rich in good quality fats.

Methods
Animals and diets. The experiment was carried out with 300 Ross 308 chicken broilers randomly allocated to 4 experimental groups, in 5 replications, 15 birds per replication. Broilers were reared under standard conditions for 42 days. They had free access to water, and were kept under a controlled light cycle. For the first 10 days, all birds were fed the same starter diet balanced to meet their nutritional demands (Table 4). On day 11 of life, they started to receive respective diets (Table 5) Antioxidant capacity. 2-Thiobarbituric acid (TBA) in tissues was determined by the extraction method according to 59 , which involved measuring the absorbance of color solution, the color of which developed as a  www.nature.com/scientificreports/ result of the reaction between fat oxidation products (mainly malonaldehyde) and TBA. Approximately 2 g of fat was weighed into a centrifuge tube to the nearest 0.01 g, to which 5 cm 3 of 10% trichloroacetic acid was added; then the mixture was triturated for 2 min with a glass rod. Next, 5 cm 3 of 0.02 molar TBA solution was added and the sample was triturated again for 2 min and centrifuged for 10 min at 4000 rpm. After centrifugation, the solution was filtered into a glass tube, and after sealing the opening with polythene sheeting, the color was developed for 24 h at room temperature. Afterwards, samples were collected for colorimetric determination. The absorbance was measured using a Hitachi U-1100 spectrophotometer at 532 nm against the reagent blank. The reagent blank was prepared by adding 5 cm 3 of 10% trichloroacetic acid and 5 cm 3 of 0.02 molar TBA solution to a glass tube. Measurements for radical scavenging activity in analyzed tissues were performed by routine assay procedure 60 using a synthetic DPPH radical (1,1-diphenyl-2-picrylhydrazyl). Folin-Ciocâlteu reagent was used as an oxidizing reagent, and all the chemicals were purchased from SIGMA-ALDRICH CHEMIE GMBH (Munich, Germany) in the highest available purity.
Glutathione (GSH) concentration was determined in tissues by means of the OXISRESEARCH BIOXYTECH GSH/GSSG-412™ test (Foster City, CA, USA). Before the analysis, the samples were frozen with the addition of M2VP (1-methyl-2-vinyl-pyridium trifluoromethanesulfonate) at a temperature of 80 °C. The released, reduced GSH was determined in accordance with the detailed instruction provided by the kit's producer. The absorbance reading (λ412) and the measurement of reaction kinetics were performed using the microplate reader Synergy 4 (BIOTEK; Winooski, VT, USA). The results were calculated using Gen5 software (BIOTEK). GSH concentration was expressed in thiol groups (mmol-SH groups).

Fatty acid composition.
Total lipids from tissues were extracted following the procedure described by  The elongase index (EI) and thioesterase index (TI) were calculated as follows 64 : The activity of Δ5-desaturase + Δ6-desaturase was determined using the following equation 65 : Estimation of health indices. The established fatty acid profile enabled calculating the n-3/n-6 ratio, PUFA/SFA ratio, monounsaturated fatty acids (MUFA)/SFA ratio, and UFA/SFA ratio. The saturation index (S/P) and the atherogenicity (AI) and thrombogenicity (TI) indices were computed as follows 47 :  Statistical evaluation. The Principal Component Analysis (PCA) was conducted for tentative data exploration using the STATISTICA 13.0 software. Mean values of fatty acid contents in the analyzed samples were processed using the PS IMAGO PRO 6.0 statistical package employing one-way analysis of variance (ANOVA). Tukey's test was used to determine the significance of differences between the examined groups. In turn, Student's t-test was used to compare two groups. The above-mentioned analyses were expected to provide an explicit answer to the question whether the use of various selenium forms in a diet positively affects the modification of fatty acid profile in selected tissues of chicken broilers. At the same time, they would enable establishing the most appropriate form of selenium to be used in broiler diet in order to improve meat quality and, thereby, to enrich the human diet with fine-quality fats.

Data availability
The datasets utilized and analyzed during this investigation are available upon reasonable request from the corresponding author.